The manner in which alloreactive T lymphocytes recognize determinants on class I H-2 molecules is not understood. Analysis of the reactivity of cytotoxic T lymphocytes (CTL) with cells transformed with hybrid ( or exon-shuffled) H-2 class I genes, has suggested that only the alpha-2 domains express determinants recognized by CTL. I have isolated a somatic cell mutant which expresses a Dd molecule that lacks a serological determinant residing in the alpha-3 domain but retains all of the alpha-1 and alpha-2 determinants. Sequencing of the alpha-3 domain of the mutant Dd molecule shows that there is a single point mutation resulting in the substitution of a lysine for the glutamic acid at position 227. In contrast to what is predicted from the exonshuffling experiments, this Dd mutant cell line is not killed by clone lines of anti-Dd reactive CTL. I therefore propose that integrity of at least some residues in the alpha-3 domain is required for CTL recognition because they contribute to a conserved (or monomorphic), determinant that, in cellular recognition, defines the molecule as a class I molecule. This application seeks to perform oligonucleotide directed mutagenesis in the alpha-3 domain of the cloned H-2Dd gene and analyze CTL recognition of cells transfected with the mutant genes. These studies should provide insights as to the role of the alpha-3 domain of class I molecules in CTL recognition.